scholarly journals Multi-stage chemical heating for instrument-free biosensing

2018 ◽  
Author(s):  
John P. Goertz ◽  
Kenya M. Colvin ◽  
Andrew B. Lippe ◽  
John L. Daristotle ◽  
Peter Kofinas ◽  
...  
Keyword(s):  
Phase Change ◽  
Rolling Circle Amplification ◽  
Melting Points ◽  
Rolling Circle ◽  
Reaction Field ◽  
Diagnostic Assays ◽  
Multi Stage ◽  
Automation Systems ◽  
Chemical Heating ◽  
Global Access

AbstractImproving the portability of diagnostic medicine is crucial to alleviating global access-to-care deficiencies. This requires not only designing devices that are small and lightweight but also autonomous and independent of electricity. Here, we present a strategy for conducting automated multi-step diagnostic assays using chemically generated, passively regulated heat. Ligation and polymerization reagents for Rolling Circle Amplification of nucleic acids are separated by melt-able phase-change partitions, thus replacing precise manual reagent additions with automated partition melting. To actuate these barriers and individually initiate the various steps of the reaction, field ration heaters exothermically generate heat in a thermos while fatty acids embedded in a carbonaceous matrix passively buffer the temperature around their melting points. Achieving multi-stage temperature profiles extends the capability of instrument-free diagnostic devices and improves the portability of reaction automation systems built around phase-change partitions.

scholarly journals Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

2018 ◽  
Vol 64 (12) ◽  
pp. 1704-1712 ◽  
Author(s):  
Felix Neumann ◽  
Iván Hernández-Neuta ◽  
Malin Grabbe ◽  
Narayanan Madaboosi ◽  
Jan Albert ◽  
...  
Keyword(s):  
Seasonal Influenza ◽  
Rolling Circle Amplification ◽  
High Specificity ◽  
Clinical Samples ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Diagnostic Assays ◽  
Padlock Probes ◽  
Probe Assay ◽  
Digital Quantification

Abstract BACKGROUND Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants—A(H1N1), A(H3N2), B/Yamagata, and B/Victoria—and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa™ Flu A/B & RSV Direct. RESULTS The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital readout for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

scholarly journals Addressing widespread misidentifications of traditional medicinal mushrooms in Sanghuangporus (Basidiomycota) through ITS barcoding and designation of reference sequences

IMA Fungus ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shan Shen ◽  
Shi-Liang Liu ◽  
Ji-Hang Jiang ◽  
Li-Wei Zhou
Keyword(s):  
Species Identification ◽  
Rolling Circle Amplification ◽  
Practical Method ◽  
Morphological Characters ◽  
Its Sequences ◽  
Commercial Development ◽  
Medicinal Mushrooms ◽  
Rolling Circle ◽  
Reference Sequences ◽  
Its Barcoding

Abstract“Sanghuang” refers to a group of important traditionally-used medicinal mushrooms belonging to the genus Sanghuangporus. In practice, species of Sanghuangporus referred to in medicinal studies and industry are now differentiated mainly by a BLAST search of GenBank with the ITS barcoding region as a query. However, inappropriately labeled ITS sequences of “Sanghuang” in GenBank restrict accurate species identification and, to some extent, the utilization of these species as medicinal resources. We examined all available 271 ITS sequences related to “Sanghuang” in GenBank including 31 newly submitted sequences from this study. Of these sequences, more than half were mislabeled so we have now corrected the corresponding species names. The mislabeled sequences mainly came from strains utilized by non-taxonomists. Based on the analyses of ITS sequences submitted by taxonomists as well as morphological characters, we separate the newly described Sanghuangporus subbaumii from S. baumii and treat S. toxicodendri as a later synonym of S. quercicola. Fourteen species of Sanghuangporus are accepted, with intraspecific distances up to 1.30% (except in S. vaninii, S. weirianus and S. zonatus) and interspecific distances above 1.30% (except between S. alpinus and S. lonicerinus, and S. baumii and S. subbaumii). To stabilize the concept of these 14 species of Sanghuangporus, their taxonomic information and reliable ITS reference sequences are provided. Moreover, ten potential diagnostic sequences are provided for Hyperbranched Rolling Circle Amplification to rapidly confirm three common commercial species, viz. S. baumii, S. sanghuang, and S. vaninii. Our results provide a practical method for ITS barcoding-based species identification of Sanghuangporus and will promote medicinal studies and commercial development from taxonomically correct material.

scholarly journals MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy

Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 222
Author(s):  
Chenxin Fang ◽  
Ping Ouyang ◽  
Yuxing Yang ◽  
Yang Qing ◽  
Jialun Han ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Rolling Circle Amplification ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Sensing Platform ◽  
Mirna Detection ◽  
Substrate Cleavage ◽  
Amplification Strategy ◽  
Circular Template ◽  
Dual Signal Amplification

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.

scholarly journals Evaluation of Immuno-Rolling Circle Amplification for Multiplex Detection and Profiling of Antigen-Specific Antibody Isotypes

2021 ◽  
Vol 93 (15) ◽  
pp. 6169-6177
Author(s):  
Sara Horta ◽  
Felix Neumann ◽  
Shu-hao Yeh ◽  
Christoffer Mattsson Langseth ◽  
Kadri Kangro ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Rolling Circle Amplification ◽  
Multiplex Detection ◽  
Rolling Circle ◽  
Antibody Isotypes
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